Molecular evolution of latrotoxins: neurotoxins from black widow spider venom

描述（由申请人提供）：黑寡妇蜘蛛（Latrodectus属）的毒液含有一种有效的神经毒素，a-latrotoxin，能引起脊椎动物神经分泌细胞大量释放递质。尽管a- latotoxin能够引起人类严重的中毒，但在脊椎动物神经递质释放的研究中也起着重要作用。一种欧亚物种（L. tredecimguttatus）毒液的生化特征表明，除了a-latrotoxin外，它还含有其他蛋白质序列与a-latrotoxin相似的神经毒素，但选择性地影响无脊椎动物。a-latrotoxin和这些无脊椎动物毒素被认为是一个共同基因家族的功能不同的成员，统称为latrotoxin。虽然有证据表明，其他latodectus（31种）及其亲缘种中也可能存在latodectus毒素，但除了L. tredecimguttatus之外，latotoxin的多样性几乎未知。本研究旨在通过描述蜘蛛毒素基因家族的多样性来研究其分子和功能进化。比较分子分析也将与a-latrotoxin的功能研究相结合，以确定导致其脊椎动物毒性的进化步骤。该研究有三个具体目标：(1)利用基因组学和蛋白质组学方法表征三种焦点物种中latrotoxin的多样性；(2)通过重组a-latrotoxin对脊椎动物细胞的功能测定，比较3种a-latrotoxin的兴奋性；(3)利用生物信息学分析方法研究懒虫毒素的全球系统发育分布和分子进化，以确定a-懒虫毒素起源的时间和环境。实现这些目标将提供关于这一具有医学意义的基因家族的基本信息，并将确定合成有害毒素的物种范围。相关性：a- latotoxin是研究脊椎动物神经递质释放的重要工具。相关毒素的表征将提供更广泛的分子探针，以更精确地确定神经元和胶质信号传导的机制基础，有助于神经发育和疾病的研究。拟议中的研究还将允许生物医学研究人员合成专门用于中和来自不同蜘蛛物种的a-latrotoxin的抗蛇毒血清；由于a-latrotoxin中毒而住院并不罕见，目前的治疗方法在治疗某些物种咬伤方面的有效性降低。

DESCRIPTION (provided by applicant): The venom of black widow spiders (genus Latrodectus) contains a potent neurotoxin, a-latrotoxin, that evokes mass release of transmitters from vertebrate neurosecretory cells. Though capable of causing severe envenomation in humans, a-latrotoxin also plays an instrumental role in the study of vertebrate neurotransmitter release. Biochemical characterization of one Eurasian species' (L. tredecimguttatus) venom shows that in addition to a-latrotoxin, it contains other neurotoxins that are similar to a-latrotoxin in protein sequence, but which selectively affect invertebrates. Together, a-latrotoxin and these invertebrate toxins are considered functionally distinct members of a common gene family collectively known as latrotoxins. While evidence suggests that latrotoxins may occur in other Latrodectus (31 species) and related species, latrotoxin diversity is virtually unknown outside of L. tredecimguttatus. The proposed study seeks to investigate the molecular and functional evolution of the latrotoxin gene family by characterizing its diversity across spider species. Comparative molecular analyses will also be coupled to functional studies of a-latrotoxin in order to identify the evolutionary steps leading to its vertebrate toxicity. The proposed study has three specific aims: (1) to characterize the diversity of latrotoxins in three focal species using genomic and proteomic approaches; (2) to compare the excitatory properties of a-latrotoxin from these three species by conducting functional assays of recombinant a-latrotoxins on vertebrate cells; and (3) to investigate the global phylogenetic distribution and molecular evolution of latrotoxins with bioinformatic analyses to pinpoint the timing and circumstances surrounding the origin of a-latrotoxin. Achieving these aims will provide fundamental information about this medically significant gene family, and will also establish the range of species that synthesize hazardous latrotoxins. Relevance: a-latrotoxin is an essential tool for the study of vertebrate neurotransmitter release. Characterization of related toxins will provide a wider breadth of molecular probes to more precisely determine the mechanistic basis of neuronal and glial signaling, aiding studies of neurological development and disorders. The proposed research will also allow biomedical researchers to synthesize antivenoms tailored to neutralize a-latrotoxin from different spider species; hospitalization due to a-latrotoxin envenomation is not uncommon and current therapies have reduced effectiveness at treating bites from certain species.